We are studying a mechanism by which Escherichia coli regulates translational initiation after an energy source shift-down. Recent evidence indicates that Initiation Factor 2 (IF2) from down-shifted cells is altered in its ability to be released from 70S ribosome-mRNA-fmet-tRNA complexes and that this results from an alteration in IF2 itself, rather than in the ribosome or any other ribosome-associated factor. We propose to determine the chemical nature of the modification made to IF2 in down-shifted cells. We will than seek to identify and purify the enzyme which catalyzes the modification and determine what, if any, physiological effectors govern the rate of modification. We also hypothesize a de-modifying enzyme and will seek to identify and purify such an enzyme, and determine whether its action is subject to physiological control. Proof that the modification governs functional activity will depend on our ability to demonstrate loss of function after in vitro modification and regain of function after in vitro de-modification. Finally, we will ascertain which element(s) of this regulatory system is altered in variant strains (Tic minus) which lack the ability to control translational initiation in vivo after shift-down.